The thymus is a bilobate organ derived from embryonic endoderm and mesoderm differentiation and is located just above the heart (reviewed in [1a]). It is the primary organ for maturation of T cells. This process involves the interaction between developing thymocytes and thymic stromal cells. Thymic stromal cells which forms the thymic architecture, have been classified according to their anatomical localization. They encompass a very diverse array of cell types, including cortical and medullar epithelial cells, fibroblasts, macrophages and dendritic cells (reviewed in [2a]). Stromal cells control the differentiation of haematopoietic precursors derived from the liver or the bone marrow into T lymphocytes: T-cell differentiation is defined by the acquisition of maturation markers such as CD4, CD8 and the T-cell receptor complex (TCR), which conditions the reactivity of immature thymocytes with thymic stromal cells. The early thymocyte progenitors entering the thymus do not express T-cell-specific molecules, such as CD3, the alpha or beta-chain of the TCR, or the CD4 and CD8 molecules. These CD4−CD8−cells, referred to as double-negative (DN) cells, then become CD4+CD8+, the so-called double-positive (DP) stage, and then progressively acquire TCR molecules. The final maturation of T-cells involves the selective loss of either the CD4 or the CD8 molecules to generate fully mature single-positive (SP) cells with cytotoxic/suppressor or helper/regulator function, respectively.
During this process, the TCR-mediated positive and negative selection of T cells ensures the selection of a diverse TCR repertoire able to react with foreign peptide presented by autologous major histocompatibility complex (MHC) molecules, but tolerant to self-antigens. This property renders the thymus an attractive site for manipulation of T-cell tolerance. To date, results on tolerance induction via direct manipulation of the thymus have been scarce (reviewed in [3a]). However, previous studies using intrathymic (IT) injection of pancreatic islet cells [4a], soluble antigens [5a] or adenoviral vectors [6a, 7a] have shown that induction of tolerance to foreign antigens in non-immunosuppressed animals is feasible. Since the production and maturation of thymocytes may be a life-long process, a major drawback to the utilisation of soluble antigens or adenoviruses is their short-term expression in the thymus [8a]. Indeed, modulation of the selection process should stop upon the disappearance of the antigen, which might be a problem for long-term tolerance induction.
Due to their ability to infect resting cells and to stably integrate into the genome, lentiviral vectors represent powerful new tools for long-term expression of a given transgene in vivo [9a]. Lentiviral vectors have been used successfully in vivo to infect hepatocytes and muscle cells [10a], antigen-presenting cells [11a, 12a], as well as cells of the central nervous system [13a].
The inventors postulate that lentiviral vectors might be better suited than adenoviral vectors for long-term IT expression of a foreign gene. They therefore investigated the pattern of infection of a ubiquitous lentiviral vector after IT injection.
Young adult mice were injected in the thymus with lentiviral vectors expressing eGFP or the hemaglutinin of the Influenza virus under the control of the ubiquitous phospho glycerite kinase promoter. Thymi were examined 5 to 90 days thereafter directly under a UV-light microscope and by flow cytometry. Intrathymic injection of lentiviral vectors predominantly results in infection of stromal cells that could be detected for at least 3 months. Importantly, hemaglutinin expression by thymic stromal cells mediated negative selection of thymocytes expressing the cognate T-cell receptor. In addition and despite the low multiplicity of infection, transduced thymocytes were also detected, even 30 days after injection.